Project Summary ? Project 1 Triple negative breast cancer (TNBC) is characterized by physical changes in the tumor microenvironment, including aberrant multiscale structure, and mechanics of the extracellular matrix (ECM), disturbed distributions of soluble factors, and population-level abnormalities in cellular composition and collective behavior (the tumor ecology). Additionally, obesity is known to increase the risk and worsen the prognosis for TNBC. However, the functional interconnections between these physical changes of the microenvironment and tumor metabolism remain unclear. This gap in understanding can be largely attributed to a lack of computational and experimental models that permit reliable prediction, recapitulation, and study of tumor and obesity-associated physical mechanisms in TNBC. By integrating biomaterials, tissue engineering, and microfabrication, our groups have made significant advances in the design of realistic culture microenvironments that recapitulate biological and physical properties of tumors. Furthermore, we have iteratively coupled these platforms with computational models to generate novel testable hypotheses. Here, we will capitalize on this expertise to investigate the overall hypothesis that physical changes in the microenvironment regulate malignancy by perturbing cellular metabolism. Furthermore, we will test whether obesity primes for tumorigenesis through similar physical and metabolic mechanisms. We will focus on hypoxia-inducible factor alpha (HIF1a) as a first candidate of the molecular pathways that underlie these effects, with other candidates pursued in collaboration with Projects 2 and 3. These hypotheses are based on our preliminary data and will be tested in 3 aims that will integrate engineering-centric approaches with transgenic mouse models, PDXs, patient-derived organoid cultures, and drug testing. Aim 1 will examine the physical mechanisms by which tumor and obesity-associated ECM induce metabolic reprogramming of mammary epithelial and stromal cells and define the consequences of these properties on malignancy. Aim 2 will define how HIF1a mechanistically links physical changes of the microenvironment with tumor metabolism, metastasis, and drug response. Aim 3 will analyze the collective cellular dynamics of tumor and stromal cell metabolic reprogramming in complex physical microenvironments. Collectively, these studies will reveal physical mechanisms in tumor metabolic reprogramming and link these changes to targetable molecular mechanisms thus generating new physical sciences-inspired insights for clinical translation. Project 1 heavily uses both the Tissue Microfabrication and Biophysics and Metabolic Imaging Cores and complements Projects 2 and 3 by testing the role of ECM physical properties in microvesicle biogenesis (Project 2) and by evaluating tumor cell migratory and invasive properties in response to defined ECM physical and transport characteristics (Project 3).